title: Nature of SLC41A1 complexes: report on the split-ubiquitin yeast two hybrid assay.
abstract: The Na(+)/Mg(2+) exchanger SLC41A1 is involved in the pathophysiology of various disease conditions. It forms high-molecular-mass, possibly hetero-oligomeric protein complexes in transgenic HEK293 cells. Therefore, we attempted to identify binding partners of SLC41A1 by utilizing the split-ubiquitin modification of the yeast two-hybrid assay. As the most prominent binding partners in our experimental system, we identified 3-beta-hydroxysteroid-Δ(8),Δ(7)-isomerase and B-cell receptor associated-protein 31. Other polytons (interactors appearing in the screen more than once) included: IER3IP1, PPIB, UPF0480 protein C15orf24, SPINT2, C14orf1/PEBP28, NIFIE14, YIPF6, and KCP2. In total, 20 polytons and 38 singletons (interactors appearing in the screen only once) were identified. The polytons identified were mostly endoplasmic reticulum-located, integral proteins involved in protein maturation, N-glycosylation, protein folding, anterograde transport of proteins, protein secretion, and the regulation of apoptosis. Among the singletons, we identified SLC31A2, SLC35B1, SLC39A13, CRACM1, and MTCH2 as putative binding partners of SLC41A1. Interestingly, we did not identify interactions among SLC41A1 molecules. Most of the identified interactors are integral proteins localized in cellular compartments other than the cytoplasmic membrane, whereas SLC41A1 is targeted to the cytoplasmic membrane where it performs its core function. None of the interactors was confirmed by mass spectrometry. Instead, we identified among the proteins co-purified with strep-tagged SLC41A1: ACCA1, UBB, ATX2L, HSP7C and TBB. We therefore conclude that: (1) identified interactors form transient rather than stable complexes with SLC41A1, (2) the molecular interactors identified primarily among the polytons might contribute to the production, proper folding, and maturation of SLC41A1 in the endoplasmic reticulum and Golgi apparatus, (3) most of the interactors identified among singletons might undergo similar maturation steps (post-translational modification), anterograde transport, and protein sorting as SLC41A1.
authors.affiliation: Institute of Veterinary-Physiology, Freie Universität Berlin, Berlin, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Nestler
authors.forename: Gerhard
authors.initials: G
authors.lastname: Sponder
authors.forename: Katrin
authors.initials: K
authors.lastname: Rutschmann
authors.forename: Lucia
authors.initials: L
authors.lastname: Mastrototaro
authors.forename: Christoph
authors.initials: C
authors.lastname: Weise
authors.forename: Juergen
authors.initials: J
authors.lastname: Vormann
authors.forename: Monika
authors.initials: M
authors.lastname: Schweigel-Röntgen
authors.forename: Martin
authors.initials: M
authors.lastname: Kolisek
mesh_terms: D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D013251:Steroid Isomerases / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D046912:Multiprotein Complexes; C476304:SLC41A1 protein, human; D025801:Ubiquitin; D013251:Steroid Isomerases
keywords: Na+/Mg2+ exchanger (NME); binding partner; mass-spectrometry (MS); polyton; singleton; split-ubiquitin yeast two-hybrid assay (SU-YTH)